--- title: "Quick check of IBDMDB metagenomics data subset" author: David Barnett date: last-modified format: html keep-md: false theme: dark: darkly light: flatly embed-resources: true toc: true toc-location: left toc-depth: 4 fig-dpi: 200 fig-width: 7.5 fig-height: 5 fig-responsive: true code-tools: true code-fold: show code-link: true --- ```{r} library (tidyverse)library (here)library (phyloseq)library (seriation)library (microViz)``` ## Intro - Focusing on 1 sample per participant fairly arbitrarily chosen as first in visit order- Checking for any overall compositional differences between Controls and UC and CD patients- Warnings about lack of counts I'll fix in microViz as they're annoying when the metaphlan profiles are proportions## Get combined data ```{r} # also fix/delete messy variables that produce variable name warnings <- read_rds (here ("data" , "phyloseq.rds" ))<- ps %>% ps_mutate (ever_bowel_surgery = ..6 ..Have.you.ever.had.bowel.surgery.,..6..Have.you.ever.had.bowel.surgery. = NULL ,diarrhea_2w = ..4 ..In.the.past.2. weeks..have.you.had.diarrhea.,hospitalised_2w = ..5 ..In.the.past.2. weeks..have.you.been.hospitalized.,diagnosis = factor (diagnosis, levels = c ("CD" , "UC" , "nonIBD" ))%>% ps_select (! contains ("In.the.past.2.weeks" )) ``` - Chuck bad samples (low reads)```{r} %>% samdat_tbl () %>% ggplot (aes (x = reads_raw)) + geom_histogram (bins = 100 ) + coord_flip () + scale_x_log10 ()``` ```{r} <- ps %>% ps_filter (reads_raw > 10 ^ 5 )``` - Compute alpha diversity```{r} <- ps %>% ps_calc_diversity (rank = "Species" , index = "shannon" , varname = "shannon_species" ``` ```{r} # take only first sample per person (by visit order) <- ps %>% ps_arrange (visit_num) %>% ps_dedupe (vars = "Participant.ID" , method = "first" )``` ## Quick plots and analyses ### Compositions - No obvious differences between groups but quite some variation to explore and discuss.```{r, fig.width=10, fig.height=10} %>% comp_barplot (tax_level = "Genus" , facet_by = "diagnosis" ,n_taxa = 13 , merge_other = FALSE , + coord_flip ()``` ```{r, fig.width=10, fig.height=10} <- distinct_palette (n = 3 , add = NA )names (cols) <- levels (samdat_tbl (ps1)$ diagnosis)%>% ps_mutate (Diagnosis = as.character (diagnosis)) %>% tax_agg (rank = "Species" ) %>% tax_sort (by = sum, trans = "compositional" ) %>% tax_transform ("clr" , rank = "Species" ) %>% comp_heatmap (taxa = 1 : 40 , taxa_side = "right" ,tax_anno = taxAnnotation (Prev. = anno_tax_prev (bar_width = 0.3 , size = grid:: unit (1 , "cm" ))sample_anno = sampleAnnotation (Diagnosis = anno_sample ("Diagnosis" ),col = list (Diagnosis = cols), border = FALSE ``` ### CLR PCA ```{r, fig.width=8, fig.height=8} %>% tax_transform ("clr" , rank = "Species" ) %>% ord_calc ("PCA" ) %>% ord_plot (colour = "diagnosis" ) + stat_ellipse (aes (colour = diagnosis))``` ```{r} # interactive # ps1 %>% # tax_transform("clr", rank = "Species") %>% # ord_calc("PCA") %>% # ord_explore() ``` ```{r, fig.width=8, fig.height=8} %>% tax_transform ("clr" , rank = "Species" ) %>% ord_calc ("PCA" ) %>% ord_plot (colour = "diagnosis" , axes = c (1 ,3 )) + stat_ellipse (aes (colour = diagnosis))``` ```{r, fig.width=8, fig.height=8} %>% tax_transform ("clr" , rank = "Species" ) %>% ord_calc ("PCA" ) %>% ord_plot (colour = "diagnosis" , axes = c (1 ,3 ), plot_taxa = 1 : 10 )``` ### PERMANOVAs - There is a difference by Aitchison distance on Species level#### Aitchison Species ```{r} %>% tax_transform ("clr" , rank = "Species" ) %>% dist_calc ("euclidean" ) %>% dist_permanova ("diagnosis" ) %>% perm_get ()``` #### Bray Curtis Genus ```{r} %>% tax_transform ("identity" , rank = "Genus" ) %>% dist_calc ("bray" ) %>% dist_permanova ("diagnosis" ) %>% perm_get ()``` #### Binary Jaccard Species ```{r} %>% tax_transform ("binary" , rank = "Species" ) %>% dist_calc ("jaccard" ) %>% dist_permanova ("diagnosis" ) %>% perm_get ()``` ### Alpha diversity #### Compare groups - timepoint 1 - By eye the diversity of some but not all patient samples is lower than controls.```{r} %>% samdat_tbl () %>% ggplot (aes (x = diagnosis, y = shannon_species)) + geom_boxplot () + geom_jitter ()``` - Colitis vs controls ```{r} %>% samdat_tbl () %>% filter (diagnosis != "CD" ) %>% :: ggbetweenstats (x = diagnosis, y = shannon_species``` - Crohn's vs controls ```{r} %>% samdat_tbl () %>% filter (diagnosis != "UC" ) %>% :: ggbetweenstats (x = diagnosis, y = shannon_species``` #### Check timepoint 1 relation to antibiotics etc? ##### Abx ```{r} %>% samdat_tbl () %>% :: ggbetweenstats (x = Antibiotics, y = shannon_species)``` ##### Diarrhea ```{r} %>% samdat_tbl () %>% :: ggbetweenstats (x = diarrhea_2w, y = shannon_species)``` ##### Hospitalised ```{r} %>% samdat_tbl () %>% :: ggbetweenstats (x = hospitalised_2w, y = shannon_species)``` ##### Bowel surgery ever ```{r} %>% samdat_tbl () %>% :: ggbetweenstats (x = ever_bowel_surgery, y = shannon_species)``` ##### Age ```{r} %>% samdat_tbl () %>% :: ggscatterstats (x = consent_age, y = shannon_species, point.args = list (mapping = aes (colour = diagnosis))``` #### Diversity over time - Just for my own interest now, because I haven't done an IBD project before- Seems like a subset of IBD patients have persistently very low diversity- Diversity is a crude measure, compositional stability would be more interesting, but I should stop now..```{r} %>% samdat_tbl () %>% add_count (name = "n_samples" , .by = Participant.ID) %>% filter (n_samples > 3 ) %>% mutate (mean_shannon = mean (shannon_species), .by = Participant.ID) %>% ggplot (aes (x = visit_num, y = shannon_species)) + geom_hline (yintercept = 1.5 , linetype = "dashed" ) + geom_line (aes (group = Participant.ID, colour = mean_shannon < 1.5 ), alpha = 0.5 ) + geom_smooth (method = "lm" , colour = "black" ) + facet_wrap (facets = vars (diagnosis)) + guides (colour = "none" ) + theme_bw ()``` ```{r} %>% samdat_tbl () %>% group_by (diagnosis, Participant.ID) %>% summarise (n = n (), mean_shannon = mean (shannon_species),sd_shannon = sd (shannon_species)%>% ungroup () %>% filter (n > 5 ) %>% ggplot (aes (colour = diagnosis, x = mean_shannon, y = sd_shannon)) + geom_vline (xintercept = 1.5 ) + geom_point (aes (shape = mean_shannon < 1.5 )) + theme_bw ()``` ```{r} %>% samdat_tbl () %>% add_count (name = "n_samples" , .by = Participant.ID) %>% filter (n_samples > 3 ) %>% mutate (mean_shannon = mean (shannon_species), .by = Participant.ID) %>% ggplot (aes (x = reorder (Participant.ID, shannon_species, FUN = mean), y = shannon_species+ geom_hline (yintercept = 1.5 ) + geom_boxplot (aes (fill = mean_shannon < 1.5 ), outliers = FALSE ) + geom_point (size = 1 ) + facet_grid (cols = vars (diagnosis), scales = "free_x" , space = "fixed" ) + theme_bw () + theme (axis.text.x = element_blank ())``` ## Session info ```{r} :: session_info ()``` 
